GOLPH3蛋白复合体促进胰腺癌生长和侵袭的分子机制

Pancreatic carcinoma is one of the most fatal cancers with an extremely poor prognosis and high mortality rates. To clarify the molecular mechanism of growth and invasion in human pancreatic carcinoma is very important for the early diagnosis and finding effective therapeutic methods.Our previous studies found that oncoprotein Golgi phosphoprotein 3(GOLPH3) was upregulated in pancreatic ductal adenocarcinoma (PDAC) lesions compared to paired adjacent noncancerous tissue, which was correlated with the prognosis of PDAC patients. Further studies indicated that overexpression of GOLPH3 promote tumor cell growth and migration, markedly up-regulate β-catenin protein levels in pancreatic cancer cells. Using BiFC (Bimolecular Fluorescence Complementation) high throughput protein interaction screening and Phosphoprotein Profiling Microarray, we found GOLPH3 interacted with cytoplasm proteins such as STIP1 and PSMD10 in pancreatic carcinoma cell lines. Overexpression of GOLPH3 could activate ERK and Wnt/β-catenin signalling. However, the exact molecular mechanism has not been elucidated. In our present project, Co-Immunoprecipitation, ELISA, Immunofluorescence, Laser capture microdissection (LCM) and Three-dimensional cell culture methods were performed in GOLPH3 overexpression or knockdown pancreatic carcinoma cell models and pancreatic carcinoma mice models, to investigate the proteins interaction between GOLPH3 and STIP1 or PSMD10, to confirm the ERK and Wnt/β-catenin signal transductional pathways regulated by GOLPH3 , to figure out the molecular mechanisms and the function of GOLPH3 in tumor cell proliferation and invasion. Moreover, we will comfirm our results in the serum and tumor tissue samples , analyse and determine the significance and relationship between GOLPH3 and STIP1 or PSMD10 in the progress of pancreatic carcinoma. We hope to explore novel useful therapeutic targets for pancreatic carcinoma treatment.

胰腺癌死亡率高,预后极差。阐明胰腺癌生长及侵袭转移的分子机制，对于早期诊断及寻找有效的治疗途径非常重要。我们的前期研究表明，癌蛋白GOLPH3表达上调，与胰腺癌的预后相关。进一步研究发现，过表达GOLPH3上调β-catenin，促进胰腺癌细胞增殖和侵袭。我们的双分子荧光互补高通量蛋白互作筛选和磷酸化蛋白芯片结果提示，GOLPH3与压力诱导的磷酸蛋白-1(STIP1)、癌锚蛋白PSMD10等胞内蛋白结合，激活 ERK和β-catenin通路，但具体机制尚待阐明。本项目将建立高表达或敲低GOLPH3的胰腺癌细胞模型及胰腺癌小鼠模型，通过免疫共沉淀、ELISA、免疫荧光、激光捕获显微切割及三维细胞培养等方法，探讨GOLPH3与STIP1、 PSMD10相互作用激活关键信号通路的分子机制，明确其促进胰腺癌细胞生长和转移的功能，并将在患者血清和组织样本中验证相关结果，为胰腺癌治疗提供新靶点。

我们前期研究发现，胰腺癌细胞高尔基复合体膜蛋白GOLPH3过表达与胰腺癌患者预后密切相关,可是GOLPH3的作用机制仍然不清楚。应用双分子荧光互补（BiFC）高通量筛选互作蛋白，GOLPH3与压力诱导的磷酸蛋白-1（STIP1）、癌锚蛋白PSMD10等多个细胞内蛋白结合。本研究的目的是通过建立敲低GOLPH3的胰腺癌细胞模型，验证GOLPH3互作蛋白并探讨癌蛋白GOLPH3作用的细胞内分子通路，探索新的治疗方法和靶点。.本研究应用免疫共沉淀证明了GOLPH3 与STIP1为相互结合蛋白，免疫荧光染色表明两蛋白定位共存在肿瘤细胞胞浆中。比较6对癌组织和癌旁组织，免疫组织化学、免疫荧光、免疫印迹检测及定量RT-PCR结果发现GOLPH3 和STIP1均在胰腺癌组织过表达。应用实时定量端粒重复扩增方法(Real-time Quantitative-telomere .repeat amplifification Q-TRAP)检测端粒酶相对活性，敲低GOLPH3后，胰腺癌细胞（PANC1和BXPC3）的端粒酶活性和hTERT表达水平下降（STIP1蛋白表达水平没有改变）。进一步研究发现，小RNA(siRNA)敲低GOLPH3后cyclin D1 和 c-Myc 的表达下调，而且敲低STIP1后也出现cyclin D1 和 c-Myc 的表达以及端粒酶活性下调。siRNA分别敲低GOLPH3/STIP1后胰腺癌细胞的增殖能力减弱。稳定表达GOLPH3shRNA的胰腺癌细胞BXPC3接种裸鼠的皮下成瘤实验中，shRNA敲低GOLPH3组的肿瘤体积比对照组明显缩小。与对照载体组相比GOLPH3shRNA组c-Myc 和hTERT 水平和相对端粒酶活性下调，免疫组化显示GOLPH3shRNA组小鼠肿瘤组织表达GOLPH3, Ki67, and Cyclin D1 蛋白表达水平显著下降, 前凋亡蛋白Bim相反。本项研究多个实验结果均提示GOLPH3通过与STIP1相互作用共同激活端粒酶、后者上调细胞周期信号分子从而促进细胞生长。.本项研究发现了癌蛋白GOLPH3上调端粒酶活性促进胰腺癌细胞生长的新通路，GOLPH3和STIP1可能成为治疗胰腺癌的新靶标。